Mechanism Of Naphthalimide Compound Inducing Tumor Cell Apoptosis

Malignant tumor is one of the main causes of human beings' death.The WHO's statistics suggests that there are 7.1 million persons dying from malignant tumor every year on average among the world population of 5 billion odd,and the number of new cases is 8.7 million,which is still increasing year by year.Therefore,the prevention and treatment of tumor has been regarded as the important subject attracting more and more attention of all the scientists in the world,and to look for the effective target spot of the effect of tumor drugs and to compound the new anti-tumor drugs has become the important subjects in the fields of cell biology and oncology as well as others.Since Lerman proposed the model that the molecule in the plate condensed-ring aromatic structure combines with DNA in the mode of intercalating in 1961,that DNA works as the target spot of anti-tumor drugs has attracted more and more attention.A large amount of research suggests that most of the drugs for DNA damage can induce the tumor cell apoptosis to different degree.The intercalator is usually inserted between the DNA base pair by means of intercalating,to make the DNA tertiary structure change, cut off the DNA,enable the covalent cross-linking with DNA single strand or double strands,or change the DNA topology structure to influence the biochemical function.For example,the activity of the RNA polymerase or enzymeⅡin topology structure will be inhibited so that the DNA could not be replicated or transcribed,and consequently the cell apoptosis is caused.As one of the DNA intercalators,Naphthalimide compound can intercalate in the DNA,inhibiting the compound of DNA and RNA synthesis,and the topoismerase as well. In view of its obvious anti-tumor effect,it has been one of the hot topics in these years. The current Naphthalimide compounds have several problems,such as the weak antineoplastic activity,high toxic and side effect,etc.Therefore,the Naphthalimide compound with strong antineoplastic activity and slighter toxic and side effect are demanded.This research adopts the MTT method and flow cytometry,taking the Hela,MCF-7, U-251 and SMMC-7721 as the models,and select double-Naphthalimide compound C8 which can effectively induce the apoptosis of tumor cells from the compounds taking eleven kinds of Naphthalimide designed and synthesized by own as the parent substance.Observe shape change and apoptosis situation of U-251 cells under the action of C8 by inverted microscope,fluorescence microscope and electron microscope; judge the toxicity of C8 and the influence on the cell membrane of U-251 by determining lactic dehydrogenase;detect the influence on the cell cycle by flow cytometry;study the apoptosis pathway of C8 inducing the apoptosis of tumor cell and relevant regulatory genes by determining the mitochondria membrane potential,the release of Cytlchrome C and activities of Caspase-9 and Caspase-3,analyzing the mRNA content of Bcl-2,Bax and p53 by real-time quantitative analysis and analyzing Bcl-2 protein expression by Western Blotting.The series of experiments can deeply research on the mechanism of C8 inducing tumor cell apoptosis,laying the foundation for further research on antineoplastic effects of compound C8 and its application in the future.Main methods and result of this research:1.Determine the cell viability with MTT colorimetric methodMake use of the MTT colorimetric method to determine the inhibition of compounds in different concentrations(0-80μM) on the MCF-7,Hela,U-251 and SMMC-7721 cell in vitro growth after the action of 12h,18h and 24h.The result shows that among the 11 kinds of compounds,C8 has the inhibition effect on all of the four kinds of cells.However, its inhibition effects on different cells are different.The other 10 kinds of compounds have some inhibition effect on the cells,but the effect is not so obvious(P＞0.05)(See Fig. 2-1,2-2,2-3,2-4,2-5,2-6,2-7,2-8,2-9,2-10,2-11 and 2-12).After the action of 12h, compared with the contrast group(0.01%DMSO),20μM C8 can obviously inhibit the cell viability of MCF-7,U-251 and SMMC-7721(p＜0.05),but its inhibition on the cell viability of Hela is not so significant(p＞0.05).After 18 h,compared with the compounds in smaller concentration,C8 can obviously inhibit the growth of foresaid cells.After 24h,the inhibition effect is more obviously than that after 18h,and the inhibition rate tends to increase with the time of culture passing.In the same period,with the concentration of C8 increasing,the cell viability gradually reduces(p＜0.05),and the obvious dependence on the dosage appears.2.Result of determination with flow cytometerUse the flow cytometer to determine the cell apoptosis of Hela,MCF-7,U-251 and SMMC-7721 after the C8 whose final concentration is 10μmol/L acting on them respectively for 18h.The result is as follows:compared with the contrast group,C8 has stronger inducement of apoptosis to MCF-7,U-251 and SMMC-7721,at the apoptosis percentage of 12.9±1.80%,23.8±2.34%and 16.1±2.84%respectively.But its effect on the Hela is weaker,only at 7.7±2.55%,which demonstrates that the compound has certain selectivity to the tumor cell.3.Use the microscope to observe the state of cell growthIn this research,we observe that compared with the contrast group,the cell grows slower and the cell density is smaller after the 18h's action of 2.5μmol/L and 5μmol/L C8,which proves that the C8 in this concentration has the inhibition on the cell growth. The morphology of U-251 changes greatly after the 18h's action of 10μmol/L C8.That is, the unsatisfactory cell spreading,cell body retraction,rough cytoplasm,a large amount of accumulation of particles in the cell,some cells not adhering to the wall,and the cell density further reduced.After the 18h's action of 20μmol/L and 40μmol/L C8,the complete contour of most of the cells is fuzzy and the phenomenon of cell apoptosis or necrosis is obvious.Thus it can be seen that with the increase of concentration of C8, inhibition of growth of U-251 intensifies.The morphological change of U-251 shows that C8 can make U-251 peel from wall by means of disturbance of cell metabolism,change of cell adhesion and reduction of adhesive power,thus inhibit the cell proliferation through the change of cell adhesion.4.Use the fluorescence microscope to observe the cell apoptosis.Different from the cell death,the cell apoptosis is a kind of programmed cell death (PCD).The characteristic change of cell apoptosis includes the cell pycnosis,volume shrinkage,karyopycnosis and cytoplasm bubble etc.The morphological change is usually deemed to be the basis to determine whether there is any cell apoptosis. Hoechst 33342 dye is easier to enter to the apoptosis cell than to the normal cells,and the PI dye can not enter to the living cell with complete cell membrane.According to these characteristics,use the two dyes Hoechst 33342 and PI to the apoptosis cell,and the normal cell,apoptosis cell and necrosis cell can be distinguished by the fluorescence microscope or the flow cytometer.This research uses the Hoechst 33342 and PI to dye the cells and observes the cells under the fluorescence inverted microscope.The following phenomena are found to the U-251 in the experiment group:cellular atrophy, karyopycnosis,chromosome accumulation,karyon cracking to fragments,apoptotic body appearance and increase of karyon fluorescence strength.5.Observe the cell ultramicrostructureObserve the ultramicrostructure of the apoptotic cells with the transmission electron microcopy,the membrane of the cells in the contrast group is complete,the cytoplasm is distributed evenly and the karyon,karyolemma and subcellular structure are all complete. In the experiment group where the 5μM and 10μM C8 act for 18h,the cell volume shrinks and the connection disappears,away from the surrounding cells;the apoptosis characteristics including the karyopycnosis,chromatin accumulation under the karyolemma and the cytoplasm bubbling etc appear;but the cell membrane keeps complete.After the 18h action of 20μM C8,the chromatin of karyon highly condenses and tends to edge.The karyolemma and core break and there are dispersed fragments and other cell content surrounding the cell.At this moment,the karyolemma is to break or has broken and the cell has been dead.6.Determination of Lactate dehydrogenase(LDH)The cytotoxicity of C8 may be one aspect of its anti-tumor mechanism.The determination of the activity of the metabolite LDH in the culture medium of tumor cell can not only determine the state of tumor cell membrane but also check the cytotoxicity of drugs.This research chooses the U-251 as the model in vitro to determine the influence of C8 on the release rate of LDH in the supernatant of culture medium of U-251.The result shows that,comparing the experiment group with the contrast group, only a minute quantity of LDH(p＞0.05) can be detected after the 2.5μM and 5μM C8 cultured with the cell for 12h,18h and 24h.The effect of C8 in the concentration over 10μM on the release of U-251 LDH starts to increase significantly.Compared with the contrast group,the viabilities of LDH in the cell sap after the action of 20μM C8 for 12h, 18h and 24h are 118.4±3.23%,153.2±4.51%and 187.6±3.56%respectively.Compared with the contrast group,the viabilities of LDH in the cell sap after the action of 40μM C8 for 12h,18h and 24h are 121.4±4.21%,182.2±2.24%and 211.3±3.81%respectively.It shows that with the increase of acting time of C8,the viability of outside LDH obviously strengthens(p＜0.05).In the meantime,with the increase of concentration of C8,the viability of outside LDH obviously strengthens(p＜0.05).Thus it can be seen that the cytotoxicity of C8 in the concentration over 10μM on the U-251 is as follows:in the same concentration,the cytotoxicity of C8 intensifies with the increase of acting time.In the same acting time,the cytotoxicity of C8 intensifies with the increase of concentration.The cytotoxicity of C8 in concentration over 10μM on the U-251 shows the obvious dependence on concentration and time.7.Influence of cell cycle distributionThe research shows that comparing the test results of the U-251 cell cycle distribution and apoptosis percentage after the 24h action of C8 in the concentration within 0-20μM with the contrast group,with the increase of C8 concentration,the cell content in G0/G1 cycle increases,decreases in S cycle,and without obvious change in the G2/M cycle,which demonstrates that C8 can make the U-251 encounter with the blockage in G0/G1 cycle.In the meantime,with the increase of C8 concentration,the "sub G1 peak" mirroring the apoptosis characteristics appears.That is,the cell content in the sub-Gl cycle gradually increases(p＜0.05),and the cells in this cycle is usually deemed to be the apoptotic cells.This shows that the C8 can induce the U-251cell apoptosis and the apoptosis rate is in positive correlation to the C8 concentration.8.Determination of mitochondria membrane potentialThe reduction of mitochondria membrane potential occurs before the appearance of the karyon apoptosis characteristics(chromatin concentration,DNA fracture).It is deemed to be the earliest event in the cascade reaction of the cell apoptosis.Once the mitochondria membrane potential collapses,the cell apoptosis will not be reversed.The experiment result suggests that the mitochondria membrane potential rises a little after 1h action of 10μM C8.Then the mitochondria membrane potential rapidly decreases, which is the most obvious within 1-6h(p＜0.05).After 6h,the decreasing speed tends to be stable(p＞0.05).The 6h action of 2.5μM and 5μM C8 can also lead to the decreasing of mitochondria membrane potential,but the decreasing amplitudes are smaller than those of 10μM C8.This demonstrates that C8 may lead to the decrease of mitochondria membrane potential of U-251 and enable the afunction of mitochondria which is the early-state characteristic of cell apoptosis.9.The release of Cytochrome CAfter 10μM C8 acts on U-251 for 4h,8h,12h,16h and 20h,adopt Western Blotting to make preliminary analysis of cell sap and Cyt C protein content in mitochondria.The results show that:the cell sap of contrast group is hardly without Cyt C;add 10μM C8 and act for 4h,the Cyt C content in the cell sap increases,but the Cyt C content in the mitochondria begins to decrease;with the prolongation of acting time,the Cyt C content in the mitochondria will further decrease,but the Cyt C content in the cell sap will gradually increase,which prove that C8 may change the permeability of mitochondria membrane and cause the Cyt C existing between inner membrane and outer membrane to dissociate from outer membrane to the cell sap.This result keeps consistent with the occurrence time of electric potential descending of mitochondria membrane.10.The activities of Caspase-9 and Caspase-3The Cytochrome C released to the cell sap can result in Caspase activation.The Caspase protein family plays the key role during the executing period of cell apoptosis, Caspase-9 and Caspase-3 are the key executing molecules and they play roles on many ways of the apoptosis signal transduction.Detect the influences of Caspase-9 and Caspase-3 activation and the results show that:compared with the contrast group,after 3-6h of application of C8(10μM),the changes of Caspase-9 and Caspase-3 are not detected,but after 12h,the Caspase-9 and Caspase-3 in U251 are obviously activated, after 24h and 48h,the activities of Caspase-9 respectively increase to 296.9±14.23% and 326.8±12.51%of the contrast group,and the activities of Caspase-3 respectively increase to 286.2±12.01%and 308.5±13.42%.2.5μM and 5μM C8 acting for 24h can make the activities of Caspase-3 increase to 138.4±4.31%and 216.3±3.87%of the contrast group,displaying obvious dose-effect relationship(p＜0.05).11.Analyze the mRNA contents of Bcl-2,Bax and p53The Bcl-2 protein family acts as the regulation and control center in the mitochondria pathway of apoptosis,in which Bcl-2 is one of the main anti-apoptosis factors in the cell and directly participates in the regulation of mitochondria membrane potential;and Bax and etc are the apoptosis promoting proteins.The apoptosis inhibition of Bcl-2 must be realized through its formation of Hetero-dimers together with the Bax.This research adopts the real-time quantitative PCR technology to determine the mRNA contents in Bcl-2 and Bax when C8 acting on U-251 in 10 h and the result shows that:compared with the contrast group,the mRNA contents of Bcl-2 and Bax in the cells of the treatment group have no obvious change(p＞0.05),which demonstrates that C8 is likely to have no obvious influence on Bcl-2 mRNA in short time.Adopt the real-time quantitative PCR technology to determine the p53 mRNA content when C8 acting on U-251 in 10 h and the result shows that:the p53 mRNA content of the experiment group is higher than that of the contrast group;the p53 expression of C8 acting in 10 h is 1.5 times above than that of C8 acting in 0 h,which demonstrates that C8 is likely to damage DNA inherent structure in the cell nucleus, causing the p53 expression.12.Analyze Bcl-2 protein expressionThe detecting result of Bcl-2 protein by the flow cytometer shows that:the contrast group of Bcl-2 protein expression is 97.8%and the experiment group of that is 31.4%, which is less than the contrast group by 66%.The result of Western Blotting also displays the same decline trend,showing that C 8 inducing U-251 cell apoptosis maybe have certain relationship with Bcl-2 protein.Conclusions of this research:1.The Naphthalimide compound C8 has inhibitory action and inducing apoptosis action on cell growth of SMMC-7721,U-251和MCF-7.2.Maybe the cell toxicity of compound C8 is one factor of its antineoplastic mechanism.3.Compound C8 can block cell cycle in G0/G1 cycle,inhibit the DNA synthesis of tumor cell in S cycle and induce the cell apoptosis of U-251.4.Compound C8 can embed in nucleus DNA,change the structure of the nucleus DNA and cause the tumor cell into apoptosis procedures. 5.This research preliminarily determines the mitochondria pathway of the compound C8 inducing the cancer cell apoptosis:cause the release of Cytochrome C from the mitochondria to the cell sap by reducing the potential of mitochondria membrane and improving the permeability of mitochondria membrane;inhibit Bcl-2 protein expression;activate Caspase cascade reaction,and finally leading to the cell apoptosis.